玻尿酸接枝非離子型界面活性劑囊泡應用於抗皮膚老化研究

隨著社會進步、醫藥發達，高齡化社會的來臨，皮膚抗老化對大部分族群而言已成為重大議題。現在市面上很多化妝品以及醫學美容宣稱可以抗老化，但其有效性有待商榷。故本研究希望可以開發具有良好經皮吸收，並能將抗老化藥物累積於皮內的藥物載體，進而達到皮膚抗老化的訴求。本實驗選用了白藜蘆醇作為抗皮膚老化之藥物，白藜蘆醇是一種非黃酮類的多酚類天然物，諸多文獻表示因其特有的抗氧化以及抑制自由基的能力，能加強皮膚的抗老化以及預防皮膚癌症等功能。但是在經皮吸收的應用上有兩個障礙:其一白藜蘆醇容易被氧化代謝，導致其生物可用性低，以及不易經皮吸收。其二因其特有結構易被光降解，由原本有效用的反式結構(trans-isomer)轉變為沒有抗氧化能力的順勢結構(cis-isomer)。以上的性質限制了白藜蘆醇的應用性，故本研究設計非離子界面活性劑囊泡(Niosome)系統來包覆白藜蘆醇來幫助其可用率以及經皮吸收等功效。非離子界面活性劑囊泡(Niosome)是由非離子界面活性劑組成的雙層膜(bilayer)囊泡(vesicle)載體。Niosome具有以下特徵:低毒性、穩定高，並有多數文獻證明其具有良好的經皮吸收之功效。玻尿酸(hyaluronic acid)具有標靶於真皮內的纖維母細胞的功能，故本研究設計將低分子量玻尿酸接枝於非離子界面活性劑Kolliphor P407，利用核磁共振光譜儀(NMR)、紅外線分光光譜儀(FT-IR)和膠體滲透層析法(GPC)確認其化學結構和分子量。研究中使用不同種類的非離子界面活性劑: Span60、Tween 80、TPGS與P407，以及與玻尿酸接枝率16%與33% 的P407，添加膽固醇(Cholesterol)穩固載體結構，並以乙醇注射法來製備包覆白藜蘆醇之非離子性界面活性劑囊泡。對於囊泡載體會進行粒徑、表面電位以及體外皮膚穿透試驗分析。最終會以老鼠纖維母細胞L929進行細胞毒性、抗氧化(抑制ROS)能力以及細胞膠原蛋白生合成之測試，分析出最合適的奈米經皮囊泡(niosome)載體組成。實驗結果顯示，由P407-LHA 33%與P407以2:1比例相互混和製備的囊泡P407/P407-LHA33% (1/2)具有較佳的皮內藥物累積量(59.99 ± 3.16 %)，製備完成的白藜蘆醇奈米載體的粒徑約莫260 nm，表面電位為-28 mV。與其他沒有玻尿酸接枝的囊泡組別相比，P407-LHA 33%之系列囊泡的細胞毒性很低，並且還有幫助纖維母細胞生長的現象，P407/P407-LHA33%(1/2)的組別於藥物濃度為50 μM時，細胞存活率213.91 ± 8.54 %。在抗氧化能力實驗以及膠原蛋白生成實驗中，與單純白藜蘆醇以及其他未以低分子量玻尿酸接合成的非離子界面活性劑載體相比，P407-LHA 33%之系列囊泡有顯著性抑制ROS與增加膠原蛋白生成的效果，其中含藥P407/P407-LHA33 %(1/2)有較佳的抗氧化以及促進膠原蛋白生成能力，於50 µM藥物濃度其ROS生成率為28.81 ± 4.22 %、膠原蛋白生成率為142.64 ± 7.44 %。動物實驗中，P407/P407-LHA33 %(1/2)囊泡對於使用12-O-tetradecanoilphorbol (TPA)刺激老化的皮膚，能有效抑制ROS生成量(41.27 ± 5.25 %)以及促進膠原蛋白生成率 (172.95 ± 2.79 %)，從外觀上也能有回復老化之皮膚，就結果而言P P407/P407-LHA33 %(1/2)是一組非常成功的抗皮膚老化之皮膚局部運輸藥物載體。

關鍵字

玻尿酸；白藜蘆醇；囊泡；經皮吸收；抗皮膚老化

並列摘要

Skin aging has taken immerging concerned as society advanced. The effectiveness of the aesthetic medicine procedure and cosmetic still open to dispute. In our study, we developed novel drug vesicles in order to deeply penetrate through the skin and stay for increased potency. In this study, Resveratrol (3,5,4′-trihydroxystilbene) is chosen as the anti-aging drug, which has been shown to acquire potential anti-aging and to inhibit events associated with tumor initiation. There’re two major disadvantages that keep blocks Resveratrol’s further application through Percutaneous absorption：(1) the high activity makes it easily be oxidized; (2) the structure specificity make it photoisomerization extensively from trans-isomer to cis, which lost the ability to anti-oxidization. Therefore, we aim to design new non-ionic surfactant-based Vesicle Niosome to clab Resveratrol to prevent Resveratrol oxidized while penetrating through the skin and raise the bioavailability. Niosomes are osmotically active, chemically stable and have long storage time. In addition, Hyaluronic acid can target the fibroblasts in the skin. We designed to conjugate low moleculer weight hyaluronic acid with Kplliphor P407, a nonionic surfactant, using Nuclear magnetic resonance (NMR), Fourier Transform Infrared Spectroscope (FT-IR) and Gel Permeation Chromatography (GPC) to confirm the chemical structure and molecule weight of synthesized P407-LHA. In this study, different surfactants were studies like Span60, Tween80, TPGS and P407. Ethanol injection method was used for prepare the niosome, and cholesterol was added to stabilize the bilayer structure of resveratrol loaded niosome. We found out that the P407-LHA has the best performance in drug encapsulation and adsorption capacity. Using mouse fibroblast cell line L929 to test cell toxicity, anti-oxidation, and the yield of collagen synthesis; after choosing the proper noisome for animal test. Take the skin collagen content, ROS producing rate and drug does under the skin as the reference for the effectiveness of anti-aging. Our results show that P407/P407-LHA33% (1/2) niosome showed the best percutaneous absorption (59.99 ± 3.16 %). The particle size of this niosome was 260 nm, with -28 mV surface voltage and low cell toxicity compare, L929 fibroblast cell viability in 50 μM of drug concentration was 213.91 ± 8.54 %. The P407/P407-LHA33% (1/2) niosome could highly reduce the ROS (28.81 ± 4.22 %) and induce the collagen amount (142.64 ± 7.44 %) in vitro L929 fibroblast cell. Moreover, in vivo tests underlined a best effectiveness of resveratrol-loaded P407/P407-LHA33% (1/2) niosome to counteract 12-O-tetradecanoilphorbol (TPA)-promoted skin aging. P407/P407-LHA33% (1/2) niosome could highly reduce the ROS (41.27 ± 5.25 %) and induce the collagen amount (172.95 ± 2.79 %). The resveratrol -loaded P407/P407-LHA33% (1/2) niosome appeared as successful topical skin delivery system for skin anti-aging

並列關鍵字

Hyaluronic acid ； Resveratrol ； Kolliphor P407 ； Niosome ； Transdermal delivery ； topical use ； Skin anti-aging

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